Substrate For Nitric Oxide Synthase Research Articles

Potential Use of L-Arginine Amino Acids towards Proliferation and Migratory Speed Rate of Human Dental Pulp Stem Cells (hDPSCs).

Inflamed pulp has a decreased amount of protein and amino acids. L-Arginine is a semi-essential amino acid, the production of which is insufficient under oxidative stress and inflammation. L-Arginine is the sole substrate for nitric oxide synthase (NOS) to produce nitric oxide (NO), which plays an important role in cell proliferation and migration. This study aims to evaluate the proliferation and migration rate of hDPSCs after L-Arginine supplementation. Serum-starved hDPSCs were divided into four groups: control: hDPSCs in Dulbecco's modified Eagle medium; hDPSCs in 300 μmol/L of the L-Arginine based culture media group; hDPSCs in 400 μmol/L of the L-Arginine based culture media group; and hDPSCs in 500 μmol/L of the L-Arginine based culture media group, which were planted in two separate 24-well-plates (5x104 cell/well) for proliferation and migration evaluation. The proliferation of all groups was measured by using a cell count test (haemacytometer and manual checker) after 24 h. The migratory speed rate of all groups was measured by using cell migration assay (scratch wound assay) after 24 h. Cell characteristics were evaluated under microscope (Inverted microscope, Zeiss®, Observer Z1, UK) that can be read through image-J® interpretation. This image J represented the measurement of migratory speed rate (nm/h) data. Statistical analysis was conducted using one-way ANOVA and post hoc Bonferroni (p<0.05) for proliferation and post hoc LSD (p<0.05) for migration (IBM SPSS Statistics Software, version 22.0). There was a statistically significant difference in hDPSC proliferation among various concentration groups of the L-Arginine based solution (300, 400 and 500 μmol/L) compared to the control group. There was a statistically significant difference in the migratory speed rate of hDPSCs in 500 μmol/L of the L-Arginine based solution group compared to lower concentrations and control group. The highest potency of hDPSC proliferation and migration was observed in the 500 μmol/L group. (EEJ-2023-01-08).

Abstract 7660: L-Arginine metabolism is of prognostic relevance in women with breast cancer, but varies considerably between subtypes in vitro - A bedside-to-bench translational approach

Abstract Availability of the semi-essential amino acid L-arginine (L-arg) is limiting for proliferation in highly proliferative tissues. L-Arg is also a substrate for nitric oxide synthases (NOS) and arginases (ARG) 1 and 2. Methylation of L-arg residues within proteins by protein L-arg methyltransferases (PRMTs) leads to asymmetric (ADMA) and symmetric dimethylarginine (SDMA). Protein L-arg methylation is an important post-translational mechanism to regulate gene expression and cell cycle control. We measured L-arg and its metabolites in a prospective cohort of patients with primary breast cancer (BC). We further measured these metabolites and expression of genes involved in L-arg metabolic pathways in cell lines representing intrinsic BC subtypes. Plasma L-arg-related metabolite concentrations from 243 women with primary BC were associated with total mortality and disease recurrence during a median follow-up of 88 (IQR, 82-93) months. MCF-7, BT-474, SK-BR-3, and MDA-MB-468 cells representing ER-positive, HER2-positive, and triple negative BC were cultured in standard conditions and compared to MCF-12A normal breast epithelial cells. The plasma (in vivo) and intracellular concentrations (in vitro) of L-arg, L-ornithine (L-orn), L-citrulline (L-cit), ADMA, and SDMA were measured by UPLC-MS/MS. mRNA expression was quantified by qRT-PCR and protein expression was analyzed by Western Blot. In vivo, the plasma concentrations of all L-arg metabolites differed significantly between BC patients and healthy controls. L-Arg and ADMA were significantly higher in patients with recurrent disease and in those who died during follow up. ADMA was significantly associated with total mortality and recurrent disease in Luminal A patients; low L-orn/L-arg ratio was associated with survival in HER2+ BC patients, and low L-cit was associated with survival in triple negative BC. With the exception of MCF-7 cells, DDAH1 and DDAH2 mRNA concentrations in vitro were significantly higher in BC cells than in MCF-12A cells (DDAH1: 32-44 fold, DDAH2: 1.7-4.2 fold; p&amp;lt;0.05). These differences were mirrored by DDAH1 and DDAH2 protein concentrations. By contrast, MCF-7 cells were characterized by low DDAH1 and DDAH2, but high PRMT4 and PRMT6 mRNA expression and high L-arg content. BT-474 cells showed high ARG2 expression and high L-arg and L-orn concentrations, and MDA-MB-468 cells had the highest L-cit/L-arg ratio. In conclusion, regulation of L-arg metabolic pathways shows a complex and differential pattern between intrinsic BC subtypes, which is mirrored in representative cell lines in vitro. ADMA is a prognostic biomarker in Luminal A breast cancer patients; its metabolizing enzyme, DDAH, is highly overexpressed in BC cells in vitro. Thus, fingerprinting of L-arg metabolism may offer novel personalized treatment options within intrinsic BC subtypes. Citation Format: Juliane Hannemann, Leticia Oliveira Ferrer, Antonia Röglin, Fiona Kleinsang, Volkmar Müller, Isabell Witzel, Rainer Böger. L-Arginine metabolism is of prognostic relevance in women with breast cancer, but varies considerably between subtypes in vitro - A bedside-to-bench translational approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7660.

Nitric Oxide as a Determinant of Human Longevity and Health Span.

The master molecular regulators and mechanisms determining longevity and health span include nitric oxide (NO) and superoxide anion radicals (SOR). L-arginine, the NO synthase (NOS) substrate, can restore a healthy ratio between the dangerous SOR and the protective NO radical to promote healthy aging. Antioxidant supplementation orchestrates protection against oxidative stress and damage-L-arginine and antioxidants such as vitamin C increase NO production and bioavailability. Uncoupling of NO generation with the appearance of SOR can be induced by asymmetric dimethylarginine (ADMA). L-arginine can displace ADMA from the site of NO formation if sufficient amounts of the amino acid are available. Antioxidants such as ascorbic acids can scavenge SOR and increase the bioavailability of NO. The topics of this review are the complex interactions of antioxidant agents with L-arginine, which determine NO bioactivity and protection against age-related degeneration.

Inhibitory effect of S-nitroso-N-acetylpenicillamine on the basolateral 10-pS Cl- channel in thick ascending limb.

We have previously reported that L-arginine, a nitric oxide synthase substrate, inhibits the basolateral 10-pS Cl- channel through the cGMP/PKG signaling pathway in the thick ascending limb (TAL). As a NO releasing agent, the effect of S-nitroso-N-acetyl-penicillamine (SNAP) on the channel activity was examined in thick ascending limb of C57BL/6 mice in the present study. SNAP inhibited the basolateral 10-pS Cl- channel in a dose-dependent manner with an IC50 value of 6.6 μM. The inhibitory effect of SNAP was abolished not only by NO scavenger (carboxy-PTIO) but also by blockers of soluble guanylate cyclase (ODQ or LY-83583), indicating that the cGMP-dependent signaling pathway is involved. Moreover, the inhibitory effect of SNAP on the channel was strongly attenuated by a protein kinase G (PKG)-specific inhibitor, KT-5823, but not by the PDE2 inhibitor, BAY-60-7550. We concluded that SNAP inhibited the basolateral 10-pS Cl- channels in the TAL through a cGMP/PKG signaling pathway. As the 10-pS Cl- channel is important for regulation of NaCl absorption along the nephron, these data suggest that SNAP might be served as a regulator to prevent high-salt absorption related diseases, such as hypertension.

Arginase 1 expression is increased during hepatic stellate cell activation and facilitates collagen synthesis.

Activation of hepatic stellate cells (HSC) is a key event in the initiation of liver fibrosis. Activated HSCs proliferate and secrete excessive amounts of extracellular matrix (ECM), disturbing liver architecture and function, leading to fibrosis and eventually cirrhosis. Collagen is the most abundant constituent of ECM and proline is the most abundant amino acid of collagen. Arginine is the precursor in the biosynthetic pathway of proline. Arginine is the exclusive substrate of both nitric oxide synthase (NOS) and arginase. NOS is an M1 (proinflammatory) marker of macrophage polarization whereas arginase-1 (Arg1) is an M2 (profibrogenic) marker of macrophage polarization. Differential expression of NOS and Arg1 has not been studied in HSCs yet. To identify the expression profile of arginine catabolic enzymes during HSC activation and to investigate their role in HSC activation, primary rat HSCs were cultured-activated for 7 days and expression of iNOS and Arg1 were investigated. Nor-NOHA was used as a specific and reversible arginase inhibitor. During HSC activation, iNOS expression decreased whereas Arg1 expression increased. Inhibition of Arg1 in activated HSCs efficiently inhibited collagen production but not cell proliferation. HSC activation is accompanied by a switch of arginine catabolism from iNOS to Arg1. Inhibition of Arg1 decreases collagen synthesis. Therefore, we conclude that Arg1 can be a therapeutic target for the inhibition of liver fibrogenesis.

Abstract 3696: L-arginine depletion causes different proliferation responses in breast cancer subtypes - Relation to gene expression profiles of L-arginine metabolic pathways

Abstract Metabolic fingerprinting provides insight into cancer biology, improving personalized therapeutic strategies. We sought to unravel responses of the molecular subtypes of breast cancer to L-arginine depletion in vitro. Availability of the semi-essential amino acid L-arginine limits protein synthesis and cell proliferation. L-Arginine is the substrate for nitric oxide synthases (NOS) and arginases (ARG), enzymes also involved in the regulation of cell proliferation. Clinical studies have provided contrasting results regarding the role of L-arginine in breast cancer.We studied the response of different breast cancer cell lines to L-arginine depletion and the molecular mechanisms underlying this effect. MCF-7, SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 cells were cultured under standard conditions and compared to MCF-12A normal breast epithelial cells. Cells were maintained in culture media with 200 mg/L (control) or 0 mg/L of L-arginine (depletion) for up to 240 h. Cell proliferation was analyzed and intracellular concentrations of L-arginine, L-ornithine, and L-citrulline were measured by UPLC-MS/MS every 24 h. mRNA expression of genes involved in L-arginine metabolism was quantified by qRT-PCR. MCF-12A control cells did not proliferate in the absence of extracellular L-arginine. Their intracellular L-arginine content dropped sharply, whilst L-ornithine and L-citrulline were only slightly reduced. By contrast, proliferation of BT-474, SK-BR-3, and MDA-MB-468 cells was unaffected by L-arginine depletion (p&amp;lt;0.05), whilst MCF-7 and MDA-MB-231 cells showed significantly higher proliferation rates in the absence of L-arginine (p&amp;lt;0.05). L-arginine concentration dropped sharply within 24 h in MCF-7 cells, but only by 25-50% in the other cell lines (p&amp;lt;0.01). L-ornithine dropped to almost zero within 24 h in both estrogen receptor positive cell lines (MCF-7 and BT-474), whilst being only slightly reduced in SK-BR-3 cells (p&amp;lt;0.01). L-ornithine was not significantly reduced in MDA-MB-231, but strongly reduced in MDA-MB-468 cells (p&amp;lt;0.05). L-citrulline showed no significant change in any of the cell lines. mRNA expression of NOS, ARG, and the L-arginine salvage pathway (ASS1 and ASL) was differentially expressed amongst the cell lines. SK-BR-3 showed high expression of the L-arginine salvage pathway; BT-474 had the highest expression levels of NOS and ARG2, and MDA-MB had high ARG2 but very low NOS expression. Gene expression patterns of MCF-12A and MCF-7 were very similar. In conclusion, metabolic fingerprinting reveals complex and differential regulation of L-arginine pathways in breast cancer cell lines. These analyses may help to understand differences in proliferation during L-arginine depletion, thus offering a perspective to further personalize treatment options within classical molecular breast cancer subtypes. Citation Format: Juliane Hannemann, Fiona Franke, Mariola Kastner, Rainer Böger. L-arginine depletion causes different proliferation responses in breast cancer subtypes - Relation to gene expression profiles of L-arginine metabolic pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3696.

Effects of Feeding with Non-Autoclaved and Autoclaved Fructose-Arginine Mixture on Stress Resistance of Drosophila Melanogaster

The diet of modern people includes fast food that leads to the development of obesity and related diseases. One of the reasons for the negative impact of such a diet is advanced glycation end products (AGEs), substances formed as a result of the interaction of amino acids with carbohydrates, especially under the influence of high temperature (the Maillard reaction). Once in the body, these substances lead to oxidative stress and inflammation, which in turn can accelerate aging process. Also, AGEs are involved in the development of metabolic syndrome, diabetes, cardiovascular disease, and cancer. However, mild oxidative stress can activate cellular defense systems and make cells more resistant to a stronger oxidative stress or other types of stress that is called hormesis or cross-tolerance. Our study shows an effect of non-autoclaved (FAMn) and autoclaved (FAMa) fructose-arginine mixtures on the body of fruit flies. In our study, we used the Drosophila melanogaster line w1118. Flies were grown on nutrient medium with the addition of different amounts of FAMn or FAMa to the final concentrations of reagents in a mixture of 10, 20 and 100 mM and maintained on the respective media until the second day of age. The flies were then used to determine physiological and biochemical parameters. The increase in absorbance at 294 nm and 420 nm and a decrease in fructose concentration in FAMa indicated that autoclaving of the fructose-arginine mixture led to caramelization of fructose and formation of Maillard products. The study showed that FAM in both forms did not affect lipid peroxide level, a marker of oxidative stress. Also, FAM in both forms did not affect the resistance of flies to hydrogen peroxide. However, FAMn, but not FAMa, increased the resistance of flies to sodium nitroprusside (SNP). This effect is likely caused by the presence of arginine, a substrate for NO-synthase, which may pre-adapt flies to •NO released from SNP. FAMn and FAMa at the concentration of 100 mM increased content of storage lipids, but decreased resistance of flies to starvation.

251 (PB-075) Poster - Plasma L-arginine metabolic profiling in breast cancer patients reflects differences in cellular gene expression and metabolic activities according to subtype – A translational study in human breast cancer

Background: L-arginine is a semi-essential amino acid; its availability for protein synthesis is limiting for cell proliferation. It is also a substrate for nitric oxide synthases (nNOS, iNOS, eNOS) and arginases (ARG) 1 and 2. The effects of L-arginine on cell proliferation and disease progression in breast cancer have remained controversial. We studied whether breast cancer subtypes show different levels of L-arginine and its metabolites, and if this relates to NOS and ARG isoform expression and tumor cell proliferation.

The possible role of nitric oxide signaling and NMDA receptors in allopurinol effect on maximal electroshock- and pentylenetetrazol-induced seizures in mice

Allopurinol, a uric-acid-lowering medication, has shown its efficacy in several studies suggesting that allopurinol can be prescribed as adjunctive cure meant for intractable epilepsy. The exact mechanism of allopurinol is still unknown. This study evaluates allopurinol’s effect on seizure threshold, seizure incidence, and mortality rate in mice models. Moreover, the possible involvement of nitric oxide (NO) pathway and N-methyl-D-aspartate (NMDA) receptors are investigated.To evaluate the effect of allopurinol on seizure, we used the pentylenetetrazole (PTZ)-induced seizure along with maximal electroshock (MES)-induced seizure. To assess the underlying mechanism behind the allopurinol activity, we used nitric oxide synthase (NOS) substrate (L-arginine), NOS inhibitors (L-NAME, aminoguanidine, 7-nitroindazole), and NMDA receptor antagonist (MK-801).Intraperitoneal allopurinol administration at a dose of 50 mg/kg in mice showed a significant (p < 0.001) anti-convulsant activity in the PTZ-induced seizure. Even though pre-treatment with L-Arginine (60 mg/kg) potentiates allopurinol’s anti-convulsant effect in the PTZ-induced seizure, pre-treatment with L-NAME (10 mg/kg), aminoguanidine (100 mg/kg), and 7-nitroindazole (30 mg/kg) reversed the anti-convulsant effect of allopurinol in the PTZ-induced seizure. In addition, pre-treatment with MK-801 also decreased the anti-convulsant effect of allopurinol in the PTZ-induced seizure. While allopurinol at a dose of 50 mg/kg and 100 mg/kg did not induce protection against seizure incidence in the MES-induced seizure, it revealed a remarkable effect in reducing the mortality rate in the MES-induced seizure.Allopurinol increases the seizure threshold in PTZ-induced seizure and enhances the survival rate in MES-induced seizure. Allopurinol exerts its anti-convulsant effect, possibly through targeting NO pathway and NMDA receptors.

ЭФФЕКТЫ ИЗОЛИРОВАННОГО И СОЧЕТАННОГО ВВЕДЕНИЯ L-АРГИНИНА И АМИНОГУАНИДИНА ПРИ ОСТРОМ ЭКСПЕРИМЕНТАЛЬНОМ ПЕРИТОНИТЕ

Objectives. To study the effects of isolated and combined administration of L-arginine and aminoguanidine in acute experimental peritonitis. Material and methods. Experiments were conducted on white male rats (n=185), divided into 5equal series, which were injected intraperitoneally, 0,6ml/100g: the 1st series (control) - 0.9%NaCl, the 2nd (experimental peritonitis, EP) - the 5th series - 15% fecal suspension, with intramuscular injection to rats of the 3rd series with: substrate of NO-synthase (NOS) - L-arginine, L-Arg (300mg/kg), 4thseries - inhibitor of inducible NOS - aminoguanidine, AG (15 mg/kg), the 5th series - L-Arg and AG in analogous doses. The signs of intoxication syndrome, changes in leukocyte differential count and peritoneal fluid, the severity of prooxidant-antioxidant imbalance, lesions of vascular endothelium and structural changes in the peritoneum were studied. Results. The research of the EP course in rats with combined administration of L-Arg and AG revealed in the presence of more significant corrective effect, in comparison with the results in EP with their isolated use, showing up in the increase of motor activity and muscular strength, normalization of breathing and thermoregulation, decrease in the severity of leukocytosis and nuclear shift of the leukogram to the left, increase in the phagocytic activity, decline in the concentration of nitrite/nitrates and malondialdehyde, increased level of reduced glutathione, decrease in the quantity of circulating endothelial cells in blood and structural disorders in peritoneum. Conclusions. The most pronounced corrective effect of combined administration of the NOS-substrate, L-Arg, and the inhibitor of its inducible isoform, AG, in rats with EP may result from the suppression of excessive NO production by AG, as well as the maintenance of the constitutive NOS-activity and various metabolic pathways in the course of L-Arg administration.

Characterization of the L-Arginine/Nitric Oxide Pathway and Oxidative Stress in Pediatric Patients with Atopic Diseases.

Introduction: L-Arginine (Arg) is a semi-essential amino acid. Constitutive and inducible nitric oxide synthase (NOS) isoforms convert Arg to nitric oxide (NO), a potent vaso- and bronchodilator with multiple biological functions. Atopic dermatitis (AD) and bronchial asthma (BA) are atopic diseases affecting many children globally. Several studies analyzed NO in airways, yet the systemic synthesis of NO in AD and BA in children with BA, AD or both is elusive. Methods: In a multicenter study, blood and urine were obtained from 130 of 302 participating children for the measurement of metabolites of the Arg/NO pathway (BA 31.5%; AD 5.4%; AD + BA 36.1%; attention deficit hyperactivity disorder (ADHD) 12.3%). In plasma and urine amino acids Arg and homoarginine (hArg), both substrates of NOS, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), both inhibitors of NOS, dimethylamine (DMA), and nitrite and nitrate, were measured by gas chromatography–mass spectrometry. Malondialdehyde (MDA) was measured in plasma and urine samples to evaluate possible effects of oxidative stress. Results: There were no differences in the Arg/NO pathway between the groups of children with different atopic diseases. In comparison to children with ADHD, children with AD, BA or AD and BA had higher plasma nitrite (p < 0.001) and nitrate (p < 0.001) concentrations, suggesting higher systemic NO synthesis in AD and BA. Urinary excretion of DMA was also higher (p = 0.028) in AD and BA compared to patients with ADHD, suggesting elevated ADMA metabolization. Discussion/Conclusion: The Arg/NO pathway is activated in atopic diseases independent of severity. Systemic NO synthesis is increased in children with an atopic disease. Plasma and urinary MDA levels did not differ between the groups, suggesting no effect of oxidative stress on the Arg/NO pathway in atopic diseases.

The development of acute experimental peritonitis with the introduction of L-arginine

High lethality in peritonitis and the data about the role of nitric monoxide in its pathogenesis, determines the feasibility of the research to study effects of the NO-synthase substrate – L-arginine. The aim of the research was to study the acute experimental peritonitis course in rats with administration of the NO-synthase substrate – L-arginine. The experiments were carried out on male rats, divided into 3 series and intraperitoneally injected with: series 1 (control) – 0,9% sodium chloride, series 2 (experimental peritonitis) – 15% fecal suspension, 0,6 ml/100 g, series 3 (EP+L-Arg) – 15% fecal suspension, 0,6 ml/100 g, followed by intramuscular administration of L-arginine, 300 mg/kg. Administration of the NO-synthase substrate – L-arginine to rats with experimental peritonitis had a corrective effect in the form of a decrease in the severity of intoxication syndrome, the reaction of leukocytes of blood and peritoneal fluid, changes in nitrites/nitrates level, a decrease in the activity of oxidative stress, endothelial dysfunction and the degree of damage to peritoneum.

Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection.

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/−ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/−ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.

In silico studies of bioactive compounds from selected African plants with inhibitory activity against nitric oxide synthase and arginase implicated in asthma

BackgroundIt is a known fact that arginine is a common substrate for arginase and nitric oxide synthase (NOS). However, an imbalance between both enzymes could lead to a change in airway responses. Reports suggest that increased activities of both enzymes could lead to airway hyper-responsiveness. Thus, the requests for NOS inhibitors that can also inhibit arginase as the elevated activities of both enzymes have detrimental consequence on airways in asthma. Bioactive compounds from Azadirachta indica, Crinum glaucum, and Mangifera indica are documented for anti-inflammatory, immunomodulatory, anti-histaminic, smooth-muscle relaxants, and anti-allergic potentials. However, the mechanisms of action of these bioactive compounds in conferring the aforementioned protections are not well characterized. The objective of this present study is to assess in silico inhibitory potentials of these bioactive compounds against NOS and arginase via binding at their active sites. The crystal structures of NOS and arginase were retrieved from the protein database, while the bioactive compounds were retrieved from PubChem. Drug-likeness of the selected bioactive compounds was assessed using DruLiTo software. The successful compounds were docked with active sites of enzymes using AutoDock Vina docking software, and the docked complexes were analyzed using LigPlot and protein-ligand profiler web server.ResultsThe findings of the study revealed that the bioactive compounds from A. indica, C. glaucum, and M. indica were able to interact with the active sites of NOS and arginase with the exception of gallic acid (from M. indica) and nimbandiol (from A. indica); these compounds showed differential binding energies (kcal/mol) and a number of them had higher binding energies than l-arginine when docked with NOS.ConclusionConclusively, the in silico analysis proposes that these compounds could prove to be probable anti-asthmatic drugs.

Effect of short-term smoking & L-arginine on coronary endothelial function assessed by cardiac magnetic resonance cold pressor testing: a pilot study

BackgroundThe effect of smoking on coronary vasomotion has been investigated in the past with various imaging techniques in both short- and long-term smokers. Additionally, coronary vasomotion has been shown to be normalized in long-term smokers by L-Arginine acting as a substrate for NO synthase, revealing the coronary endothelium as the major site of abnormal vasomotor response. Aim of the prospective cohort study was to investigate coronary vasomotion of young healthy short-term smokers via magnetic resonance cold pressor test with and without the administration of L-Arginine and compare obtained results with the ones from nonsmokers.MethodsMyocardial blood flow (MBF) was quantified with first-pass perfusion MRI on a 1.5 T scanner in healthy short-term smokers (N = 10, age: 25.0 ± 2.8 years, 5.0 ± 2.9 pack years) and nonsmokers (N = 10, age: 34.3 ± 13.6) both at rest and during cold pressor test (CPT). Smokers underwent an additional examination after administration of L-Arginine within a median of 7 days of the naïve examination.ResultsMBF at rest turned out to be 0.77 ± 0.30 (smokers with no L-Arginine; mean ± standard deviation), 0.66 ± 0.21 (smokers L-Arginine) and 0.84 ± 0.08 (nonsmokers). Values under CPT were 1.21 ± 0.42 (smokers no L-Arginine), 1.09 ± 0.35 (smokers L-Arginine) and 1.63 ± 0.33 (nonsmokers). In all groups, MBF was significantly increased under CPT compared to the corresponding rest examination (p < 0.05 in all cases). Additionally, MBF under CPT was significantly different between the smokers and the nonsmokers (p = 0.002). MBF at rest was significantly different between the smokers when L-Arginine was given and the nonsmokers (p = 0.035).ConclusionShort-term smokers showed a reduced response to cold both with and without the administration of L-Arginine. However, absolute MBF values under CPT were lower compared to nonsmokers independently of L-Arginine administration.

Multi-omic signatures of atherogenic dyslipidaemia: pre-clinical target identification and validation in humans

BackgroundDyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental model of dyslipidaemia and in patients with familial hypercholesterolemia (FH).MethodsLiquid chromatography/mass spectrometry (LC/MS) and immunoassays were used to find out blood alterations at metabolite and protein levels in dyslipidaemic ApoE−/−/LDLR−/− mice and in FH patients to evaluate their human relevance.ResultsWe identified 15 metabolites (inhibitors and substrates of nitric oxide synthase (NOS), low-molecular-weight antioxidants (glutamine, taurine), homocysteine, methionine, 1-methylnicotinamide, alanine and hydroxyproline) and 9 proteins (C-reactive protein, proprotein convertase subtilisin/kexin type 9, apolipoprotein C-III, soluble intercellular adhesion molecule-1, angiotensinogen, paraoxonase-1, fetuin-B, vitamin K-dependent protein S and biglycan) that differentiated FH patients from healthy controls. Most of these changes were consistently found in dyslipidaemic mice and were further amplified if mice were fed an atherogenic (Western or low-carbohydrate, high-protein) diet.ConclusionsThe alterations highlighted the involvement of an immune-inflammatory response system, oxidative stress, hyper-coagulation and impairment in the vascular function/regenerative capacity in response to dyslipidaemia that may also be directly engaged in development of atherosclerosis. Our study further identified potential biomarkers for an increased risk of atherosclerosis that may aid in clinical diagnosis or in the personalized treatment.

Метаболизм L-аргинина у гипертензивных крыс при угнетении аргиназы L-норвалином

The hypotensive effect of arginase inhibition in the experiment [1] can be realized in two ways: by relaxation of blood vessels by an increase in the concentration of nitric oxide (NO) and/or a decrease in the amount of circulating fluid. The first effect occurs with an increase in the availability of the substrate of NO synthase (NOS) - arginine (ARG). The second - with an increase in diuresis. To clarify the ways in which arginase affects the regulation of blood pressure (BP) we have analyzed the metabolic characteristics of the amino acid L-arginine in hyper- and normotonic rats during inhibition of arginase activity by the administration of its inhibitor L-norvaline. The concentration of arginine and its metabolite ornithine in blood, urine, and in the homogenate of kidney tissue was measured using high-performance liquid chromatography with the separation on a reversed-phase sorbent and with a fluorescence detection. Norvaline was injected intraperitoneally (ip) once a day at a dose of 30 mg/kg for 7 days. In experiments under the influence of norvaline, no significant increase in the concentration of amino acids in the blood plasma was found either in hypertensive rats (ISIAH strain) or in normotensive control (WAG strain). The concentration of arginine in the urine of ISIAH animals treated with norvaline was found doubled, while in WAG rats it did not change. The daily excretion of arginine under norvaline increased in normotonics only slightly, and in hypertensive rats it increased almost threefold. At the same time, norvaline increased diuresis only in hypertensive animals. Under the influence of norvaline, the content of arginine per 1 g of kidney weight increased almost twofold in hypertensive rats, while in normotensive rats it did not change. The studies performed allow us to conclude, that inhibition of arginase by norvaline had a stronger hypotensive effect in hypertensive rats than in normotensive rats. At the same time, the hypotensive effect of norvaline in normotonic rats can be provided by the nitric oxide system, whereas in hypertensive rats this function is taken over by diuresis.

The restorative effect of apocynin and catalase in l-arginine induced hypotension on normotensive subjects - the role of oxidative stress.

L-arginine is a substrate for nitric oxide synthase (NOS) responsible for the production of NO. This investigation studied the effect of apocynin, an NADPH oxidase inhibitor and catalase, an H2O2 scavenger on L-arginine induced oxidative stress and hypotension. Forty Wistar-Kyoto rats were treated for 14 days with vehicle, L-arginine (12.5mg/ml p.o.), L-arginine+apocynin (2.5mmol/L p.o.), L-arginine+catalase (10000U/kg/day i.p.) and L-arginine plus apocynin+catalase respectively. Weekly renal functional and hemodynamic parameters were measured and kidneys harvested at the end of the study for histopathological and renal NADPH oxidase 4 (Nox4) assessments. L-arginine administration in normotensive rats decreased systolic blood pressure (120±2 vs 91±2mmHg) and heart rate (298±21 vs 254±15b/min), enhanced urinary output (21.5±4.2 vs 32±1.9ml/24h , increased creatinine clearance (1.72±0.56 vs 2.62±0.40ml/min/kg), and fractional sodium excretion (0.88±0.16 vs 1.18±0.16 %), caused proteinuria (28.10±1.93 vs 35.26±1.69mg/kg/day) and a significant decrease in renal cortical blood perfusion (292±3 vs 258±5bpu) and pulse wave velocity (3.72±0.20 vs 2.84±0.13m/s) (all P<0.05). L-arginine increased plasma malondialdehyde (by ~206 % P<0.05) and NO (by~51 %, P<0.05) but decreased superoxide dismutase (by~31 %, P<0.05) and total antioxidant capacity (by~35 %, P<0.05) compared to control. Renal Nox4 mRNA activity was approximately 2.1 fold higher (P<0.05) in the L-arginine treated rats but was normalized by apocynin and apocynin plus catalase treatment. Administration of apocynin and catalase, but not catalase alone to rats fed L-arginine, restored the deranged renal function and structure, prevented hypotension and enhanced the antioxidant capacity and suppressed Nox4 expression. These findings suggest that apocynin and catalase might be used prophylactically in states of oxidative stress.

Infusion therapy for cardiovascular diseases: the allowed limits

Background. Analysis of the mortality structure of patients with coronavirus disease (COVID-19) had found that 69.2 % of non-survivors had hypertension. Comorbid diabetes mellitus (31.8 %) and coronary heart disease (28.2 %) were also common. During pandemic, it is necessary to maintain optimal cardiovascular therapy by continuing to administer its main drugs (acetylsalicylic acid, statins, β-blockers, angiotensin-converting enzyme inhibitors – ACEI).&#x0D; Objective. To describe infusion therapy (IT) for cerebrovascular and cardiovascular diseases in settings of the COVID-19 pandemic.&#x0D; Materials and methods. Analysis of the literature on this topic.&#x0D; Results and discussion. Although the spike proteins of the new coronavirus have the tropism to ACE-2, discontinuation of ACEI is unwarranted and may worsen the course of cardiovascular disease (CVD). Particular attention should be paid to the diagnosis of acute coronary syndrome (ACS) in COVID-19. In myocardial infarction, myocarditis or cardiomyopathy on the background of COVID-19, there is a moderate increase in troponin, brain natriuretic peptide and N-terminal pro-B-type natriuretic peptide. An increase in D-dimers is a prognostic marker of the unfavorable prognosis. The algorithm for the ACS diagnosis includes the detection of typical clinical symptoms, ECG analysis, detection of disorders of local contractility of the left ventricle. Determination of troponin in patients without clinical manifestations of ACS with nonspecific manifestations of COVID-19 is not recommended. As for reperfusion therapy strategies, it is indicated in patients with symptoms of ischemia lasting &gt;12 hours and a persistent increase in ST in two adjacent leads. In the absence of prior testing for coronavirus infection, all patients should be managed according to the tactics for COVID-positive patients. In non-STEMI, patients should be stratified according to their risk level (very high, high, moderate, low). In case of high risk, the early (&lt;24 hours) invasive strategy is reasonable, in case of intermediate risk it is reasonable to consider noninvasive treatment. It should be remembered that the use of certain drugs for the treatment of COVID-19 (azithromycin, chloroquine, hydroxychloroquine, lopinavir, ritonavir) is associated with a risk of cardiotoxicity and life-threatening arrhythmias. Cardiotoxicity monitoring (determination of the corrected QT interval) should be performed before the start of therapy and then once in 5 days, primarily in risk groups (men &gt;55 years, women &gt;65 years and people with the CVD history). Lopinavir and ritonavir may also decrease the levels of active metabolites of clopidogrel and increase – of ticagrelor, so prasugrel is the antiplatelet drug of choice for COVID-19. Amiodarone also interacts with a large number of antiviral drugs. In turn, statins have multiple immunomodulatory effects including increase of the innate antiviral immune response. It is recommended to continue taking those statins that were prescribed earlier. If co-administration with lopinavir and ritonavir is required, the minimum dose of rosuvastatin or atorvastatin should be started. These antivirals are able to interact with calcium channel blockers and increase their concentration, so the dose of amlodipine and diltiazem can be reduced by 50 %. Endothelial dysfunction (ED) caused by a viral infection leads to the excessive thrombin formation and inhibition of fibrinolysis, increasing the risk of thrombotic complications. Nitric oxide (NO) plays an important role in counteracting ED. NO also inhibits the replication of the acute severe respiratory syndrome coronavirus and improves the survival of infected cells. L-arginine (Tivortin, “Yuria-Pharm”) is the only substrate for NO synthase that catalyzes the formation of NO in endothelial cells. According to the results of the own study, Tivortin helped to reduce the content of fibrinogen and soluble fibrin-monomer complexes, as well as to increase the thromboplastin time. Endothelium-dependent vasodilation also improved after administration of Tivortin. Tivorel (“Yuria-Pharm”) contains L-arginine and L-carnitine, which allows this drug to increase the survival of cardiomyocytes and endothelial cells, to restore homeostasis in the affected areas of the myocardium, and to counteract the progression of atherogenesis and thrombosis. In case of COVID-19, it is also advisable to prescribe edaravone (Ksavron, “Yuria-Pharm”), which neutralizes the cytokine storm, inhibits lipid peroxidation, protects against endothelial damage and, penetrating the blood-brain barrier, counteracts cerebral edema. In case of the need in IT, it is advisable to choose Reosorbilact (“Yuria-Pharm”), which has anti-shock, rheological, detoxifying, alkalizing and osmodiuretic effects. In hypovolemic shock and intracranial hemorrhage, the use of isotonic low-molecular-weight gelatin preparations (Volutenz, “Yuria-Pharm”) has been shown.&#x0D; Conclusions. 1. In the absence of prior testing for coronavirus infection, all patients should be managed following the tactics for COVID-positive patients. 2. The use of azithromycin, chloroquine, hydroxychloroquine, lopinavir, ritonavir is associated with a risk of cardiotoxicity and life-threatening arrhythmias. 3. ED, caused by a viral infection, increases the risk of thrombotic complications. 4. It is reasonable to include the required solutions (Tivortin, Tivorel, Ksavron, Reosorbilact, Volutenz) into the combined IT of COVID-19 patients.

Some Aspects of Role of Nitric Oxide in the Mechanisms of Hypertension (Experimental Study).

BackgroundModulation of endothelial function is a therapeutic option to reduce some of the significant complications of hypertension. However, the relationship between endothelial dysfunction reduced nitric oxide (NO) production, and the development of hypertension is not fully understood. To establish a potential pathogenetic link between impaired NO synthesis and hypertension, we investigated the results of competitive interaction of the substrate of NO synthase, L-arginine, and its analog, an non-selective inhibitor of NO synthase, N-nitro-methyl ether-L-arginine (L-NAME), in experimental rats.MethodsArterial hypertension was induced in male Wistar rats by intraperitoneal administration of L-NAME (Sigma-Aldrich) for 4 - 7 weeks. During the last 3 weeks, to a separate group of animals simultaneously with L-NAME, L-arginine (Sigma-Aldrich) was administered. In animals monitored for systolic and diastolic pressure, the level of NO in blood samples was determined spectrophotometrically using a Griess reagent.ResultsAdministration of L-NAME for 4 - 7 weeks induced an irreversible decrease of NO content in blood, a reversible increase of systolic pressure (SP) and diastolic pressure (DP), and an irreversible increase in pulse pressure (PP). In rats against the background of 7 weeks of intraperitoneal administration of L-NAME, during the last 3 weeks, they were injected with L-arginine, the SP and DP indices returned to their initial values, PP decreased and the NO content in arterial blood increased.ConclusionsThe results of the study indicate the presence of residual endothelial dysfunction (characterized by insufficient NO) after the correction of hypertension. Therefore, in developing the new therapeutic approaches for the treatment of hypertension, it is necessary to include drugs that, in addition to correcting blood pressure, will support normalization, and potentiation of endothelial function and endogenous NO synthesis.